L-methionine γ-lyase (EC 4.4.1.11) is an enzyme which requires pyridoxal 5′-phosphate (PLP) as a coenzyme and catalyzes α, γ-dissociation and γ-substitution of L-methionine or its derivatives and also α, β-dissociation and β-substitution of S-substituted L-Cysteine or its derivatives [Tanaka, H. et al., Biochemistry, 16, 100–106 (1977)]. This enzyme has been isolated and purified mainly from Pseudomonas putida and its physicochemical and enzymological properties have already been investigated [Nakayama, T. et al., Anal. Biochem., 138, 421–424 (1984)]. Some researches have reported the mechanism of enzymatic reaction catalyzed by L-methionine γ-lyase [Esaki, N. et al., FEBS Lett. 84, 309–312 (1977); Nakayama, T. et al., Biochemistry, 27, 1587–1591 (1988)]. L-Methionine γ-lyase comprises a tetramer of homogeneous subunit (monomer) and one molecular of coenzyme PLP binds to a subunit [Nakayama, T. et al., Biochemistry, 27, 1587–1591 (1988)]. These references are related to enzymes derived from natural source. Further, reported is the preparation of a recombinant enzyme by means of genetic engineering [Inoue, H. et al., J. Biochem., 117, 1120–1125 (1995) and the like, sequence No. 1]. However, the reference does not describe or suggest a functionally modified L-methionine γ-lyase, which is improved in the stability.
It has been reported that the wild type L-methionine γ-lyase purified from a culture of P. putida has an anti-tumor activity [WO94/11535, Publication date, May 26, 1994]. Furthermore, It has been suggested that L-methionine γ-lyase may be therapeutic agents for obesity [Orentreich, N et al.; J. Nutr., 123, 265–274 (1993)], Parkinson's disease [Crowell, Jr., et al.; Behav. Neur. Biol., 59, 186–193 (1993)], cardiovascular diseases [Lockwood, B. C. et al.; Biochem. J., Z279, 675–682 (1991)] or aging [Orentreich, N., et al.; J. Nutr., 123, 269–274 (1993)]; [Hoffman, R. N.; Heaman Cell, 10, 69–80 (1997)]. The amino acid sequence and the encoding DNA of L-methionine γ-lyase have been also disclosed [Inoue, et al.; J. Biochem., 117, 1120–1125 (1995)]. The half life of L-methionine γ-lyase in blood of an animal e.g. rat is short, 80 min and extends to only about double even by using a polyethylene glycol modification [Tan, et al.; Protein Expression Purif., 12, 45–52 (1998)]. In this case, the stability of the enzyme may be improved by chemical modification with polyethylene glycol at the Lys residues exposed on the surface of the enzyme. However, the chemical modification at an amino acid residue is completely different from an introduction of mutation. Therefore, the success about the increase of the structural stability of L-methionine γ-lyase with polyethylene glycol does not suggest a success in the increase of the structural stability of L-methionine γ-lyase by substitution, insertion or deletion of the amino acid residues. Furthermore, the chemical modification by means of a stabilizer of protein such as polyethylene glycol and the like did not suggest the further increase of the structural stability of functionally modified L-methionine γ-lyase.
In general, the does of a drug useful for medicines should be small. Accordingly, the supply of a functionally modified L-methionine γ-lyase with an enhanced activity and a long half-time, by means of introduction of a mutation such as substitution of amino acid residue, contributes to the development of a useful anti-tumor agent and the like. The increase in half-life of functionally modified L-methionine γ-lyase is expected by chemical modification with polyethylene glycol and the like. Enhancement of an anti-tumor activity and a reduction of side effects are expected by combining the functionally modified L-methionine γ-lyase and other anti-tumor agents.